Conifer oleoresin is a mixture of turpentine and rosin that functions in insect defense and in wound sealing (Johnson, M. & Croteau, R. (1987) in Ecology and Metabolism of Plant Lipids (Fuller, G. & Nes, W. D., eds) pp 76-91, ACS Symposium Series 325, American Chemical Society, Washington, D.C.; Gijzen, M., et al., (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., et al., eds) pp 8-22, ACS Symposium Series 525, American Chemical Society, Washington, D.C.). Turpentine is composed of monoterpene (C.sub.10) and sesquiterpene (C.sub.15) olefins, while rosin is composed of diterpene (C.sub.20) resin acids (FIG. 1). The volatile turpentine fraction of conifer oleoresin, which may consist of up to 30 different monoterpenes (Lewinsohn, E., et al., (1993) Phytochem. Anal. 4, 220-225) and an even larger number of sesquiterpenes (See Example 1, herein) furnishes a solvent for the diterpene resin acids which, upon stem wounding, harden to form a mechanical barrier thereby sealing the wound site (Johnson, M. A. & Croteau, R. (1987) in Ecology and Metabolism of Plant Lipids eds. Fuller, G. & Nes, W. D. (Am. Chem. Soc., Washington, D.C.), ACS Symp. Series 325, pp. 67-91).
Grand fir (Abies grandis) has been developed as a model system for the study of both constitutive and wound-induced oleoresin formation (oleoresinosis). The composition of the monoterpene olefin and the diterpene resin acid fractions of grand fir oleoresin has been defined (Lewinsohn, E., et al., (1993) Phylochem. Anal. 4, 220-225), and the induced biosynthesis of these natural products upon stem wounding has been described in detail (Gijzen, M., et al., (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., et al.. eds) pp 8-22, ACS Symposium Series 525, American Chemical Society, Washington, D.C.; Lewinsohn, E., et al., (1992) in Regulation of Isopentenoid Metabolism (Nes, W. D., et al., eds) pp 8-17, ACS Symposium Series 497, American Chemical Society, Washington, D.C.; Gijzen, M., et al., (1992) Arch. Biochem. Biophys. 294, 670-674; Funk, C., et al., (1994) Plant Physiol. 106, 999-1005). The time-course of induction, after wounding, of the monoterpene synthases involved in turpentine formation has been analyzed by immunoblotting techniques and the process of induced oleoresinosis was thus shown to involve de nova synthesis of these enzymes (Gijzen, M., et al., (1992) Arch. Biochem. Biophys. 294, 670-674). The cDNA sequence of abietadiene synthase, a diterpene cyclase from grand fir that is involved in resin acid biosynthesis (LaFever, R. E., et al., (1994) Arch. Biochem. Biophys. 313, 139-149)) has been reported (Stofer Vogel, B., et al., (1996) J. Biol. Chem. 271, 23262-23268), and several cDNA clones encoding monoterpene synthases from this conifer species have recently become available (Bohlmann, J., et al., (1997) J. Biol. Chem. 272, 21784-21792).
In comparison with the monoterpenes and diterpenes of conifer oleoresin, the sesquiterpenes of conifer turpentine have received relatively little experimental attention because they constitute less than 10% of the oleoresin. The relatively low concentrations of sesquiterpenes in conifer oleoresin may, however, belie their biological significance. Sesquiterpenoid phytoalexins, i.e., antibiotic compounds, are well known in angiosperm species (Threlfall, D. R. & Whitehead, I. M. (1991) in Ecological Chemistry and Biochemistry of Plant Terpenoids (Harborne, J B. & Tomas-Barberan, F. A., eds) pp 159-208, Clarendon Press, Oxford, UK), suggesting that the sesquiterpenes of conifer oleoresin may play a similar role in antibiosis.
A conifer oleoresin sesquiterpene that has been relatively well-characterized is juvabione. Juvabione is the methylester of todomatuic acid, an oxygenated derivative of the sesquiterpene olefin bisabolene (FIG. 2). The conifer sesquiterpene juvabione resembles insect juvenile hormones and, thus, can disrupt insect development and reproduction at metamorphosis and diapause (Bowers, W. S., et al., (1976) Science 193, 542-547; Bowers, W. S. (1991) in Herbivores: Their Interaction with Secondary Plant Metabeolites, Vol. I, G. A. Rosenthal and M. R. Berenbaum, eds. (Acad. Press, San Diego), pp. 431-456). Juvabione is sometimes referred to as "paper factor" because its presence in paper made from trees of the genus Abies inhibits maturation of insect larvae reared in contact with the paper (Slama, K. & Williams, C. M. (1965) Proc. Natl. Acad. Sci. USA 54, 411-414; Slama, K. & Williams, C. M. (1966) Nature 210, 329-330; Bowers, W. S., et al., (1966) Science 154, 1020-1021). Accumulation of todomatuic acid, the precursor of juvabione, in grand fir after insect feeding suggests that biosynthesis of the juvenile hormone analogue is induced de novo in response to insect attack (Puritch, G. S. & Nijholt, W. W. (1974) Can. J. Bot. 52, 585-587).
Only a single sesquiterpene synthase, E-.beta.-farnesene synthase, from a gymnosperm source, maritime pine (Pinits pinaister), has been reported (Salin, F., et al., (1995) J. Plant Physiol. 146, 203-209). In contrast, several sesquiterpene synthases from angiosperms have been described (Dehal, S. S. & Croteau, R. (1988) Arch. Biochem. Biophys. 261, 346-356; Munck, S. L. & Croteau, R. (1990) Arch. Biochem. Biophys. 282, 58-64; Belingheri, L., et al., (1992) Plant Sci. 84, 129-136), and a number of genes encoding sesquiterpene synthases involved in phytoalexin biosynthesis in angiosperms have been isolated (Facchini, P. J. & Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89, 11088-11092; Back, K. & Chappell, J. (1995) J. Biol. Chem. 270, 7375-7381; Chen, X.-Y., et al., (1995) Arch. Biochem. Biophys. 324, 255-266).